Quantifying Axon Growth in Complex Environments Project Summary: Our long-term objective is twofold: to elucidate the cellular and molecular mechanisms that underlie axon guidance after injury, and to develop biomaterial platforms to support and enhance axon growth. Our working hypothesis is that the combination of multiple growth-promoting cues will enable axon growth to overcome the local inhibitory environment (i.e., glial scar) that develops post-injury. To test this hypothesis will require the fabrication of a new physical platform upon which to study neuronal growth. The platform will (1) deliver a combination of growth-promoting cues in a controllable and quantifiable manner;and (2) provide a means by which to test a stimulatory environment against an inhibitory environment. These platforms will make possible innovative experiments that will test for the first time how combinations of guidance cues promote axon growth in an inhibitory environment. Relevant to NIBIB's mission to improve health by promoting fundamental discoveries, design, and development in bioengineering, our objective is to correlate axon growth and direction to specific quantities and ratios of stimulatory and inhibitory cues, thus establishing the basis for new strategies for nerve regeneration. This innovative multidisciplinary proposal combines the complimentary expertise of the Principal Investigator in neuronal development, regeneration, and biomaterials and the Co-Investigator in electrochemistry, microfabrication, and surface characterization, to fabricate a platform capable of delivering precise quantities of both biological guidance cues and electrical stimulation (Aim 1). Aim 2 focuses on determining if specific stimulatory guidance cues (i.e., electrical stimulation, laminin-1, and nerve growth factor) are synergistic at enhancing neurite growth. Aim 3 focuses on determining if specific stimulatory guidance cues can promote neurite growth to overcome an inhibitory environment (i.e., chondroitin sulfate proteoglycans). With pharmacological inhibitors and laser inactivation approaches, we will determine how integrin and trk receptors and downstream kinases that converge on the microtubule cytoskeleton function to interpret multiple guidance cues. Results from these studies will advance the field of biomaterials for nerve regeneration by providing more comprehensive knowledge of the requirements for axon growth in complex environments. PUBLIC HEALTH RELEVANCE: Nerves fail to regenerate after injury and current medical practice is unable to manipulate effectively the process of nerve regeneration. The proposed research seeks to solve this problem by quantifying how guidance cues, both individually and in combination, promote axon growth in an inhibitory environment such as a nerve injury site.